SHUTTLE MUTAGENESIS - A MINI-TRANSPOSON FOR PRODUCING PHOA FUSIONS WITH EXPORTED PROTEINS IN NEISSERIA-GONORRHOEAE

Shuttle mutagenesis is a method for producing stable mini-transposon (mTn) insertions into the genome of Neisseria gonorrhoeae (gonococcus, Gel and other microbes. Using an mTn3 derivative, we have produced an mTn (mTnCmPhoA) which contains a phoA' gene lacking its N-terminal signal sequence useful for isolating genes which encode exported proteins. mTnCmPhoA was characterized in Gc and Escherichia coli using a cloned target containing the Gc genes, opaE1, pilA and pilB. PhoA(+) Gc containing pilB::mTnCmPhoA insertions confirm that PilB is an exported protein in Gc. This system will be useful for isolating and characterizing extracytoplasmic virulence factors from Gc and other bacterial pathogens.